Expression and purification of avian influenza virus H5N1 NP protein, and screening interaction proteins of host cells in vitro
10.3760/cma.j.issn.1003-9279.2010.01.010
- VernacularTitle:H5N1-NP蛋白的原核表达及与宿主蛋白相互作用的初步研究
- Author:
Nai-Fu WANG
1
;
Jing MA
;
Xiao-Guang ZHANG
;
Xiao-Mei ZHANG
;
Tian BAI
;
Min WANG
;
Le-Ying WEN
;
Da-Yan WANG
;
Yue-Long SHU
;
Ling ZHOU
;
Yi ZENG
Author Information
1. 天津出入境检验检疫局动植食检测中心
- Keywords:
Influenza A virus;
Nucleoproteins;
Proteininteraction mapping
- From:
Chinese Journal of Experimental and Clinical Virology
2010;24(1):27-29
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express and purify H5N1 influenza virus (A/Anhui/1/2005) NP in prokaryotic system and to explore the NP-interacting proteins of human bronchial epithelial cells BEAS-2B in vitro . Methods The full length H5N1 NP gene fragment was amplified by PCR, inserted into prokaryotic expression vector(pET30a) to generate NP expression plasmid pET30a-NP. After transforming pET30a-NP into E. coli (BL21), the expression of soluble NP protein was induced by IPTG. The expressed NP protein was purified by two steps with metal chelation chromatography and ion exchange chromatography. Then the total proteins of BEAS-2B cells was extracted for screening the components which have protein-protein interaction with purified NP by pull-down and LC-MS/MS methods. Results The expression of H5N1 NP protein could be induced by IPTG in bacterial system using expression plasmid pET30a-NP. The soluble NP was purified. Twenty proteins were found by pull-down and LC-MS/MS, the further experiments may be needed to prove protein-protein interaction between them. Conclusion The soluble H5N1 NP fusion protein with high purity was obtained and twenty proteins were found which could interact with it by pull-down and LC-MS/MS.
